Method of inducing an immune response using vaccinia virus recombinants encoding GM-CSF

ABSTRACT

A method of inducing expression of immune active cytokines in tumors in situ is provided wherein a vaccinia virus vector capable of inducing expression of a selected cytokine is generated and injected into a tumor so that cells of the tumor express the selected cytokine. A method of enhancing immunity in a host by administration of a vaccinia virus vector is also provided. Methods of treating cancer by administration of these vaccinia virus vectors are also provided.

This application is the U.S. National Phase of PCT/US95/05968, filedNov. 5, 1995, which is a continuation-in-part of U.S. application Ser.No. 08/242,268, filed May 13, 1994, pending.

BACKGROUND OF THE INVENTION

Numerous attempts have been made to modulate a host's immune system as ameans for treating cancers. Such attempts include: active immunotherapyusing tumor or tumor antigen containing vaccines or immune activelymphokines; adoptive immunotherapy using a host's peripheral blood ortumor infiltrating lymphocytes expanded in culture and reinjected;passive immunotherapy by administration of monoclonal antibodies; andlocalized immunotherapy using intralesional administration of agentssuch as Bacillus Calmette-Guerin (BCG). The most effective of theseapproaches has been localized therapy with BCG for melanoma metastasisto the skin and superficial bladder cancer. While the mechanism ofaction of BCG is not completely understood, studies clearly show thatsuccessful immunotherapy of this type is associated with recruitment ofT cells to the tumor.

Cytokines such as the interleukins are important mediators incell-mediated immune responses in a host. The cell-mediated immuneresponse ("local immune responses") is produced by thymus derivedlymphocytes or T-cells. T-cells detect the presence of invadingpathogens through a recognition system referred to as the T-cell antigenreceptor. Upon detection of an antigen, T-cells direct the release ofmultiple T-cell lymphokines including, but not limited to, theinterleukin-2 family (IL-2). IL-2 is a T-cell growth factor whichpromotes the production of many more T-cells sensitive to the particularantigen. This production constitutes a clone of T-cells. The sensitizedT-cells attach to cells containing the antigen. T-cells carry out avariety of regulatory and defense functions and play a central role inimmunologic responses. When stimulated to produce a cell-mediated immuneresponse, some T-cells respond by acting as killer cells, killing thehost's own cells when these have become infected with virus and possiblywhen they become cancerous and therefore foreign. Some T-cells respondby stimulating B cells while other T-cells respond by suppressing immuneresponses.

Examples of other interleukins which are mediators in cell-mediatedimmune responses include interferon-γ (IFN-γ), granulocyte-macrophagecolony stimulating factor (GM-CSF), interleukin-4 (IL-4), interleukin-5(IL-5) and interleukin-12 (IL-12). IFN-γ activates macrophages andenhances expression of immune-reactive antigens on tumor cells. GM-CSFactivates macrophages and stimulates macrophage and dendrite cellrecruitment and differentiation. IL-4 is a T cell derived helperlymphokine which participates in the regulation of growth anddifferentiation of B and T cells. IL-5 is a T cell derived lymphokinewhich has its primary effects on the expansion of eosinophils. There isevidence which suggests that eosinophils, when recruited to a tumorsite, may have direct anti-tumor effects. IL-12 is a heterodimericlymphokine initially purified from the conditioned medium of a human Blymphoblastoid cell line. Murine IL-12 has now been cloned andexpressed. IL-12 stimulation has been shown to enhance antigenpresentation and the cytolytic activity of natural killer cells.

The value of cytokine-based gene therapy was suggested in preclinicalmurine studies. Inoculation of mice with experimental tumors transfectedwith genes for tumor necrosis factor (Asher AL, et al., J. Immunol. 1991146:3227), interleukin-2 (Fearon ER, et al., Cell 1990 60:397), and IL-4(Golumbek PT, et al., Science 1991 254:713) resulted in growth andsubsequent rejection of the injected tumor. In many cases the mice wereshown to generate a systemic anti-tumor response. IL-4 transfectedtumors regressed and lead to the regression of admixed non-transfectedtumors in mice (Tepper PI, et al., Cell 1989 57:503). This immunotherapywas also effective in nu/nu mice demonstrating a non-T cell componentwhich may contribute to localized therapy. IL-4 transfected RENCA cellshave been shown to generate specific T cell immunity to the tumor, andresult in elimination of pre-existing non-local tumor growth (GolumbekPT, et al., Science 1991 254:713).

Current approaches to this form of therapy involve the growth andstabile gene modification of tumor cells to produce cytokines, theirexpansion in vitro, and reinjection into the host. While this type oftherapy may be feasible in experimental systems, the lack of ability togrow the majority of tumors in vitro, the requirements for in vitrogenetic modification of each patient's tumor, and the reinjection ofviable tumor into the patient limit the clinical applicability of theapproach.

It has now been found that expression of immune active cytokines intumors can be induced in situ by administration of a vaccinia virusvector. These vaccinia virus vectors can be administered to animalssuffering from cancer as a treatment. The vaccinia virus vectors of thepresent invention are also useful in enhancing immunity to parasites andother invading pathogens which alone fail to invoke an effective hostimmune response.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of inducingexpression of immune active cytokines in tumors in situ which comprisesgenerating a vaccinia virus vector capable of inducing expression of aselected cytokine and injecting the vaccinia virus vector into a tumorso that cells of the tumor express the selected cytokine.

Another object of the present invention is to provide a method ofenhancing immunity in a host which comprises generating a vaccinia virusvector capable of inducing expression of a selected cytokine andinjecting the vaccinia virus vector into a host so that cells of thehost express the selected cytokine.

A final object of the present invention is to provide a method oftreating cancer which comprises administering to an animal sufferingfrom cancer an amount of a vaccinia virus vector capable of inducing animmune response to the cancer in the animal.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a bar graph showing systemic immunity resulting fromintravesical instillation of the vaccinia virus vector (VAC). Micereceived intravesical instillation of VAC at 10, 100 and 1,000 plaqueforming units (pfu). Two weeks later, mice spleens were removed andtested for their ability to lyse VAC-infected MB-49 cells.

DETAILED DESCRIPTION OF THE INVENTION

It has been clearly demonstrated in a number of studies that generationof effective T-cell specific immunity can result in the elimination oftumors. In vitro transduced cytokine and viral genes expressed by tumorshave resulted in the elimination of transfected tumors and enhanced Tcell mediated immunity to non-transduced tumors. Expression of immuneaccessory molecules such as B7.1 and B7.2 has also been demonstrated toenhance anti-tumor immunity. However, in vitro manipulations of tumorsto express selected molecules has its limitations, particularly in theclinical setting. Genetic modification of tumors for cellular vaccinesis dependent upon and limited by the ability to resect and to grow eachpatient's tumor in vitro and reinjection of viable, modified tumor.

A method has now been developed for in vivo gene delivery of a genewhich expresses an immune active cytokine which obviates the need for invitro manipulations of tumor cells thus enhancing the clinicalapplicability of this therapeutic approach. In the present invention, amethod of inducing expression of immune active cytokines in tumors insitu is provided which comprises generating a vaccinia virus vectorcapable of inducing expression of a selected cytokine and injecting thevaccinia virus vector into a tumor so that cells of the tumor producethe selected cytokine. By the term "inducing" or "induces" it is meantthat the level of expression of the cytokine is measurable by methodswell known in the art and that the level of expression of the cytokineresults in an immune response. By the term "immune active cytokine" or"selected cytokine" it is meant to refer to any cytokine associated withan immune response leading to tumor destruction. Examples of suchcytokines include, but are not limited to, interferon-γ (IFN-γ),granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2(IL-2), interleukin-4(IL-4), interleukin-5 (IL-5), and interleukin-12(IL-12). The vaccinia virus vector may further comprise a gene for animmune accessory molecule such as B7.1 or B7.2. By "immune accessorymolecule" it is meant a molecule which in conjunction with the immuneactive cytokine can make the tumor more immunogenic. Unlike in vitromethods of gene transfer, infection and transfection using recombinantvaccinia has been found to be a simple, rapid and highly efficientprocedure. Vaccinia recombinants can efficiently deliver antigens to theclass I presentation pathway and have been proposed as feasible vectorsfor expressing protective antigens for vaccine delivery. Moss B andFlexner C., "Vaccinia virus expression vector", Ann. Rev. Immunol. 19875:305-324. The potential utility of vaccinia recombinants forintravesical gene therapy aimed at enhancing the immunogenicity ofbladder tumor cells was suggested by Lee SS, et al. Proc. Am. Assoc.Cancer March 1993 34:337. It has now been found that these viral vectorscan be used in a method of stimulating the immune system by inducingexpression of cytokines at a tumor site.

Vaccinia virus, a double stranded DNA poxvirus, has been wellcharacterized since its successful use as a live vaccine to preventsmallpox. As a versatile eukaryotic expression vector, vaccinia viruscan be genetically constructed to contain large fragments of foreign DNA(up to 25 kd) which have no effect on viral replication. Immunizationwith recombinant vaccinia can induce protective responses to the foreigngene(s) expressed. In the present invention a vaccinia virus vector(VAC) capable of inducing expression of a selected gene is generated inaccordance with methods well known in the art. The vaccinia virus vectormay further comprise genes encoding immune accessory molecules which inconjunction with the immune active cytokine can make the tumor moreimmunogenic.

In a preferred embodiment, a foreign gene of interest, preferably a genefor a selected cytokine, more preferably the gene for IFN-γ, GM-CSF,IL-4, IL-5 or IL-12, is first placed behind a promoter, preferably a VACpromoter, in a plasmid that can be inserted into the VAC genome byhomologous recombination. Other genes which may be incorporated into thevector include, but are not limited to, genes encoding immune accessorymolecules such as B7.1 and B7.2, or genes which inhibit IL-10production. It has recently been found that both human melanoma andbladder cancer produce the immunosuppressive cytokine IL-10. Thus,inhibition of this cytokine is believed to enhance the immunogenicity oftumors. Inhibition of the expression of IL-10 has been demonstratedthrough the use of antisense oligonucleotides complementary to the IL-10DNA or mRNA in other cells. The ability to express an antisenseoligonucleotide complementary to the IL-10 DNA or mRNA can beincorporated into vaccinia virus vectors of the present invention toinhibit IL-10 production in tumor cells, thus enhancing theimmunogenicity of these tumors.

Successful insertion of the selected gene in the plasmid is confirmed byexploiting the high transfectability of certain cell lines followingvaccinia infection. After 30 minutes of exposure to wild-type vacciniaat a multiplicity of 10:1, mouse L929 cells are transfected with aplasmid DNA-lipofectin (Gibco/BRL, Bethesda, Md.) mixture. Within hoursof transfection, abundant amounts of gene product can be observed, witha majority of the cells expressing the protein. The generation of thedesired gene can be detected using standard immunodetection techniquessuch as immunoprecipitation of metabolically-labeled proteins or westernclot of cell-extracts. Further, supernatants from theinfected/transfected cells are tested for biological activitiesassociated with the various cytokines or other proteins. Afterconfirmation that the gene of interest has been correctly inserted andencodes a biologically active protein, the plasmid is recombined in theVAC genome. The plasmids are designed such that the gene of interest isinserted between the up- and downstream halves of the VAC thymidinekinase gene. Following infection of CV-1 monkey kidney cells withnon-recombinant virus, the plasmid is delivered using calcium phosphateprecipitation. In a portion of the cells, the plasmid recombines intothe vaccinia genome, disrupting the thymidine kinase gene. The resultingrecombinants are then selected from wild-type by growth in thymidinekinase negative 143B human osteosarcoma cells in the presence ofbromodeoxyuridine. It is preferred that the Wyeth strain of vaccinia,available from the Centers for Disease Control in Atlanta, Ga. (CDC) beused as this strain was used for small pox vaccinations in the UnitedStates. However, attenuated strains of vaccinia may also be used ifimmunogenicity following attenuation is not significantly compromised.

Susceptibility of cells to the vaccinia virus was demonstrated in invitro experiments in both murine and human tumor cells. Both type ofcells were infected/transfected by vaccinia recombinants. Significantinfection/transfection of established tumors in mice was also observedfollowing intravesical administration. Systemic immunity to vaccinia didnot inhibit tumor transfection by intravesically instilled vacciniarecombinants.

The safety and maintained function of the viral gene over repeatedadministrations have also been demonstrated in humans. Five patientswith dermal, subcutaneous and/or lymph node metastases from cutaneousmelanoma were vaccinated with wild-type vaccinia virus and, four dayslater, began intratumoral injections of the same vaccine. Escalatingdoses of up to 10⁷ pfu were safely administered repeatedly with onlylocal and mild systemic reactions. Four of the patients developedanti-vaccinia virus antibody titers ≧1/3200. With rising antibodytiters, local and systemic reactions decreased. One patient with a largeexophytic lesion experienced dramatic tumor regression with multipleinjections of 10⁷ pfu of virus. Sequential biopsies of this lesion overa two month period demonstrated repeated infection over successfulproduction of viral gene protein (E3L) despite anti-viral antibodytiters as high as 1/12,800. This time interval is adequate to allowgeneration of anti-tumor immunity. It is believed that a vectorcomprising a cytokine gene would function similarly and mediate animmunoadjuvant effect.

The vaccinia virus vectors of the present invention can also be used toenhance immunity in a host. In the present invention methods ofenhancing immunity in a host are provided which comprise generating avaccinia virus vector capable of inducing expression of a selectedcytokine and injecting the vaccinia virus vector into a host so thatcells of the host express the selected cytokine. By "host" it is meantto include, but is not limited to, mammals, fish, amphibians, reptiles,birds, marsupials, and most preferably, humans. This method is alsouseful in enhancing a host's immune response to parasites and otherinvading pathogens which alone may not invoke an immune response.

In addition, the vaccinia virus vectors of the present invention can beused to mediate cytokine gene transfer into tumors with resultantproduction of soluble product. For example, a recombinant vaccinia viruscontaining the murine GM-CSF gene under the control of the early/lateP7.5 vaccinia promoter (VV-GM) was constructed. VV-GM infected murinemelanoma (B16.F10) and bladder (MB49) tumors were shown to produce highlevels of biologically active cytokine as determined by propagation ofbone marrow CFU-GM and by ELISA assay. Significant levels of GM-CSF werefound in the supernatant as soon as 6 hours following infection. Thisincreased cytokine secretion of the tumor cells can lead to tumorspecific immunity and therapeutic anti-tumor effects.

Accordingly, the vectors and methods of the present invention are usefulin the treatment of cancer. Methods of treating cancer are providedcomprising administering to an animal suffering from cancer an amount ofa vaccinia virus vector capable of inducing an immune response to thecancer in the animal. In a preferred embodiment, the vaccinia virusvector used comprises at least one gene for expression of a cytokine,preferably the gene for IFN-γ, GM-CSF, IL-4, IL-5 or IL-12. In thistreatment, the vaccinia virus vector is placed in contact with the tumorin situ either by intravesical administration or by direct injectioninto the tumor. Therefore, this method is especially useful in treatingcancers such as bladder cancer, head cancer, neck cancer, melanoma, andother cancers which grow as accessible masses and are amenable to theseroutes of administration.

The susceptibility of human prostatic carcinoma cells to vaccinia wasalso examined utilizing a recombinant vector encoding the humaninfluenza hemagglutinin antigen HA. In vitro exposure of the prostaticcell lines LNCAP and PC3 to the virus followed by immunohistochemicalstaining of the encoded HA protein demonstrated a high efficiency intumor infection/transfection. Thus, the vaccinia virus vectors of thepresent invention can also be used in the localized therapy of prostatecancer.

The vaccinia virus vectors of the present invention are administered ina vaccine formulation comprising an effective concentration of vacciniavirus vector and a pharmaceutically acceptable carrier. By "effectiveconcentration" it is meant an amount of vaccinia virus vector which whenadministered to a tumor results in measurable expression of the selectedcytokine and an enhanced immune response. Such amounts can be routinelydetermined by one of skill in the art in accordance with thisdisclosure. Pharmaceutically acceptable carriers include, but are notlimited to saline solutions and buffered solutions. Suitablepharmaceutically acceptable carriers are well known in the art and aredescribed for example in Gennaro, Alfonso, Ed., Remington'sPharmaceutical Sciences, 18th Edition 1990, Mack Publishing Co., Easton,Pa., a standard reference text in this field. Pharmaceutical carriersmay be selected in accordance with the intended route of administrationand the standard pharmaceutical practice. The vaccine formulation mayfurther comprise an adjuvant. Adjuvants are substances which are addedto therapeutic or prophylactic agents, for example vaccines or antigensused for immunization, to stimulate the immune response. Use ofadjuvants in vaccines to enhance an immune response is well known in theart.

The present invention is further illustrated by the followingnonlimiting examples.

EXAMPLES Example 1 Recombinant Vaccinia Virus

Recombinant vaccinia viruses H1-VAC and NP-VAC expressing thehemagglutinin (H1) and nucleoprotein (NP) genes derived from influenzavirus A/PR8/34 were used. Expression of both influenza polypeptides isunder the control of the early/late 7.5 K promoter. Viral stocksquantitated in pfu were maintained in BSS/BSA at -70° C. until use.

Example 2 Cell Lines

The transitional cell carcinoma (TCC) cell lines MB-49 of C57BL/6origin, MBT-2 of C3H origin and the human T24 bladder carcinoma and H1human melanoma were used.

Example 3 Antibodies, Reagents and Staining

Supernatants from hybridoma cell lines specific for the influenza Ahemagglutinin (H28-E23) and nucleoprotein antigens (HB65) were used tostain cells and tissues. The virus infected bladder tumor cells andbladder urothelium sections were fixed with cold acetone and blockedwith 0.1% fetal calf serum. HA and NP were detected with primary mouseantibody and biotin labeled antimouse IgG as the second antibody plusavidin-horseradish peroxidase (HRP) and 3.3 DAB substrate (Sigma, St.Louis, Mo.) or avidin-biotin-complex method (ABC-AP) plus alkalinephosphatase with Fast red substrate (Vector Laboratories, Inc.,Burlingame, Calif.). Tissue sections were counterstained withhematoxylin. In addition, hematoxylin-eosin (H&E) stained sections wereprepared.

Example 4 In vitro Assessment of Viral Infection and Transfection

Cells (2×10⁶) from each cell line described in Example 2 were platedinto a 24 well flat bottom plate (Fisher, Pittsburgh, Pa.). Plates wereincubated overnight, washed with phosphate buffered saline (PBS) andinfected with H1-VAC or NP-VAC (10 pfu/cell) in BSS/BSA by incubating at37° C., 9% CO₂ for 1 hour with rocking every 15 minutes. Virus wasaspirated, media was added and the plate were incubated for another 4hours. The cells were fixed with 1:1 acetone:methanol for 1 minute andwashed with PBS before immunohistochemical staining. Uninfected andrecombinant virus infected L929 fibroblasts, which are known to besusceptible to vaccinia virus infection, were used as a negative andpositive control, respectively.

The murine MBT-2 and MB-49 TCC cells were infected in vitro with H1-VAC.When compared to uninfected tumor cells, immunohistochemical stainingwith specific antibodies showed positive expression for encoded HA or NPantigens indicated by the cytoplasmic staining of virus infected TCCcells. In addition, the human bladder rumor cell line T24 and a humanmelanoma line were similarly infected in vitro.

Example 5 In vivo Assessment of Virus Infection and Transfection

Female mice, 4-6 weeks of age, were purchased from the JacksonLaboratory, Bar Harbor, Me. The mice were intravesically instilled withrecombinant vaccinia virus. Mice were anesthetized, catheterized via theurethra, then cauterized with a cautery wire (Birtcher Hyfricator, ElMonte, Calif.) by applying a single 1 second pulse at 1 watt. Afterremoval of the cautery wire, the bladders were instilled with 10⁴ MB49cells to establish intravesical growth of a tumor or either 10, 100 or1,000 pfu of vaccinia virus recombinants in PBS. At 8 and 22 hoursfollowing instillation, mice were sacrificed and bladders were removedand frozen in OCT media (Fisher) in liquid nitrogen. Bladder sampleswere stored at -70° C. until sectioned.

Mice, pre-immunized intraperitoneally with wild-type WR vaccinia (10⁷pfu), were implanted intravesically with MB-49 tumor cells. Two weeksfollowing tumor development, a single intravesical instillation ofNP-VAC (2×10⁶ pfu, shown not to have systemic toxicity in preimmunemice) was given. At 8 and 22 hours post-instillation, bladders wereremoved, sectioned and stained. In vivo expression of encoded NP wasdemonstrated at 22 hours after instillation. Similar results were seenat the 8 hour time point.

Example 6 Cytotoxic T Lymphocyte Analysis

Cytotoxic T Lymphocyte (CTL) responses to intravesical infection byvaccinia recombinants were determined by a 4 hour ⁵¹ Cr assay. Spleensof virus infected mice were isolated at 2 weeks post-intravesicalinstillation, restimulated in vitro with live virus infected syngeneicspleen stimulators (3:1) and cultured for 7 days at 37° C., 5% CO₂. Theresponder cells were assayed for cytotoxicity on ⁵¹ Cr labeled vacciniavirus infected MB49 tumor targets at effector to target ratiosindicated. Percent specific lysis was calculated as follows: (cpmexperimental release-cpm spontaneous release)/(total release-spontaneousrelease)×100. Spleens of intravesically infected C57BL/6 mice weretested for antigen specific killing of vaccinia virus infected MB-49bladder tumor target cells in 4 hour chromium release assays. Novirus-induced target lysis was seen in the 4 hour assay andvirus-specific CTL did not lyse uninfected targets. As shown in FIG. 1,concentrations as low as 10 pfu intravesically were sufficient to inducea systemic anti-vaccinia CTL response. When the dose of intravesicalvaccinia was titrated, concentrations of greater than 10⁵ pfu per mousewere lethal to nonimmunized mice, which died within 5-6 dayspost-instillation. In contrast, mice receiving a single intravesicalconcentration less than 10⁵ pfu appeared normal and survived greaterthan 2 weeks post-instillation. Mice made preimmune with anintraperitoneal injection of wild-type WR vaccinia virus (10⁷ pfu)demonstrated no morbidity at intravesical concentrations as high as2×10⁶ pfu of vaccinia recombinants per mouse.

C57BL/6 female mice were given a single intravesical instillation withvaccinia recombinant H1-VAC or NP-VAC (10⁴ pfu) to confirm infection ofthe urothelium. The mice were sacrificed, post instillation, and theirbladders were recovered for sectioning and staining. Analysis of thebladder wall by routine pathology procedures using H & E stained slidesdemonstrated that urothelial cells lining the bladder lumen were virusinfected as indicated by characteristic morphologic changes includingcell enlargement, nuclear and cytoplasmic vacuolization, as well asatypical chromatin pattern.

Example 7 Human Study Using Intratumoral Vaccinia Injections as a Vectorfor Gene Transfer

Patients in this study each had histologically documented, surgicallyincurable melanoma with at least one dermal, subcutaneous or lymph nodemetastasis which was evaluable for local response and accessible forinjection. Eligible patients were fully ambulatory with or without minortumor related symptoms, had a life expectancy of six or more months andwere at least four weeks since surgery (requiring general anesthesia)and eight weeks since chemotherapy or radiation therapy. All patientswere immunocompetent as demonstrated by one or more positive cutaneousdelayed-type hypersensitivity reactions to recall microbial antigens orto dinitrofluorobenzene after sensitization.

Patients were administered Dryvax (Wyeth-Ayerst Laboratories,Philadelphia, Pa.) supplied by the Center for Disease Control (Atlanta,Ga.) in a lyophilized state. When reconstituted as directed, theresultant product contains 25 million pfu in a volume of 0.25 ml.

Each patient was vaccinated, using a standard multipuncture method witha bifurcated vaccination needle, on the skin of the deltoid area whichin all cases was a tumor free extremity with intact regional lymphnodes. The vaccination site was evaluated visually on day 4 to confirmthat a major local reaction (erythematous papule with vesiculation andpustule formation) was in progress. Tumor treatment commenced on day 4.Dermal, subcutaneous and/or lymph node metastases were infiltrated withwild-type vaccinia virus by intralesional injection using a 25 gaugeneedle (volume of injection ranged from 0.05 to 0.1 ml). Treatment wasrepeated approximately twice weekly.

Regression of injected and uninjected lesions was judged by visualinspection and/or ultrasonography. Ultrasonography was performed using a10.0 MHz linear probe (Advanced Technology Laboratories, Inc., Bothel,Wash.) with direct contact scanning of the surface of the mass as wellas scanning with a stand-off pad (Parker Laboratories, Inc., Orange,N.J.). All masses were imaged in the sagittal and axial planes. Tumorlocation, depth of penetration and sonographic textural appearance weredetermined. Tumors were measured in millimeters (mm), with the sagittal(S) and anteroposterior (AP) dimensions taken from the sagittal imagewith the greatest dimension. The tumor width (W) was obtained from thetransverse plane. Lesional response were categorized as complete (noclinically evident residual tumor), partial (≧50% reduction in tumorvolume) or none (all others).

Punch biopsies were also performed using conventional steriledermatologic techniques. One half of the material was fixed in formalin,paraffin embedded and sections stained with hematoxylin and eosin forroutine histology. The remaining tissue was halved again fortransmission electron microscopy (EM) and immunohistochemical analysis.Tissue was either fixed in 2% glutaraldehyde or embedded in OCT (FisherScientific, Pittsburgh, Pa.) and snap frozen using liquid nitrogen.Frozen tissue was subsequently sectioned at 5 microns thickness with acryostat, fixed in cold acetone, blocked with PBS with 5% fetal calfserum (FCS) and stained with the antibody TW2.3 which is specific for anearly gene product of vaccinia virus replication (E3L). As E3L is anon-structural viral protein, positive antibody staining is indicativeof active infection.

To measure serum titers for anti-vaccinia virus antibody, ninety-sixwell plates were coated with a 10 μg/ml protein extract obtained fromcultures of human melanoma cell lines infected for 6 hours with theWyeth strain of vaccinia virus. Following blocking with PBS plus FCS,dilution series of patient sera pre- and post-immunization were added tothe wells, incubated for two hours and the plates washed. Serumanti-vaccinia virus antibodies were visualized using a peroxidaselabeled anti-human IgG heavy and light chain second reagent andorthophenyldiamine substrate. Titers were read as the reciprocal serumdilution yielding 50% maximum absorbance in the assay.

Example 8 Intralesional Infection of Human Melanoma Cells by VacciniaVirus

One patient, a sixty-five year old white female, was first diagnosedwith a primary melanoma (1 mm, level 4) of the right calf with satellitelesions in 1983. The primary lesion was excised and the dermalsatellites successfully treated with intratumoral BCG. The patient didwell until 1992 when two dermal/sc lesions appeared on the calf andfailed to respond to intratumoral BCG, systemic R24 or chemotherapy.Vaccinia treatment was initiated with a standard immunization (250,000pfu topically, 15 punctures). On day 4 of treatment, when it wasdetermined that a take was clearly in progress, intralesional vacciniawas commenced. A single metastatic lesion was injected 19 times over 88elapsed days with a total of 14×10⁷ pfu (Wyeth). Several biopsies showedprogressively intense infiltration of the tumor with lymphocytes andtumor regression. EM and immunohistological staining for vaccinia geneproducts showed successful viral infection of tumor cells in thepresence of substantial anti-vaccinia antibody titers.

Example 9 Clinical Trials

Patients are administered small pox vaccine (Dryvax, Wyeth Laboratories,by scarification. Immunity to the vaccine is demonstrated by a majorreaction characterized by pustule formation at the vaccination site andthe detection of circulating anti-vaccinia antibody. Patients exhibitingboth response are eligible for localized treatment with the cytokineproducing vaccinia vector.

Patients are treated with increasing doses of the vaccinia over aseveral week period by local (intratumoral or topical such asintravesical) administration. In the case of melanoma, head and neck,and other tumors which grow as accessible solid masses at the primaryand or metastatic sites, the vaccinia is injected into the tumor using asyringe and needle. In the case of bladder cancer, the vaccinia isinstilled onto the bladder (intravesically) using a catheter.

Patients are observed at frequent intervals for signs of toxicity, andtumor response is gauged by measuring the injected and non-injectedtumor masses for signs of shrinkage by direct visualization orradiologic (ultrasound, X-ray, MRI, etc.) methods.

Evidence of systemic antitumor immunity will be determined using invitro techniques which measure the direct interaction of lymphocytes andtumor cells. Measurements of antitumor immunity are readily accomplishedby persons with skill in this field.

What is claimed is:
 1. A method of expressing granulocyte-macrophagecolony stimulating factor in tumors in situ comprising: a) generating avaccinia virus vector encoding a gene for granulocyte-macrophage colonystimulating factor operably linked to transcriptional regulatoryelements; and b) injecting said vaccinia virus into a tumor so thatcells of the tumor express granulocyte-macrophage colony stimulatingfactor.
 2. A method of producing regression of a tumor in a mammalhaving cancer comprising administering to the tumor in said mammal avaccinia virus vector encoding a gene for granulocyte-macrophage colonystimulating factor operably linked to transcriptional regulatoryelements in an amount capable of inducing an immune response to thetumor in the mammal such that the tumor regresses.
 3. The method ofclaim 2 wherein the animal has cancer comprising bladder cancer, headcancer, neck cancer or melanoma.
 4. The method of claim 2 wherein theanimal has prostate cancer.